During many types of infections, pathogenic bacteria form multicellular communities called biofilms. In these aggregates, consumption and limited diffusion leads to steep gradients of substrate availability. Microenvironments are established that differ significantly from the chemistries of traditional laboratory liquid cultures. As bactera in biofilms respond to these conditions, the community becomes metabolically heterogeneous and exhibits increased resistance to environmental perturbations and antibiotic treatment. Although the metabolic states of bacteria in biofilms are known to be important for their recalcitrance, many questions remain regarding the principles that underlie their response to substrate limitation. We employ a colony morphology assay to study biofilm development in the prevalent nosocomial pathogen Pseudomonas aeruginosa. We modulate the availability of oxygen and nitrate, known respiratory substrates for this bacterium, and alter the ability of P. aeruginosa to produce phenazines, endogenous pigments that can also act as electron acceptors. We have observed that electron acceptor availability is a major determinant of biofilm structure. These studies suggest that colony wrinkling is an adaptation that allows P. aeruginosa cells to access oxygen through an increased surface area when other electron acceptors are not available. Measurement of the NADH/NAD+ ratio in the wild type and a mutant unable to produce phenazines has indicated that the intracellular redox state is a signal that triggers the morphotypic switch from smooth to wrinkled. Proteomic studies and genetic screens have uncovered candidate regulators, including PAS domain proteins and regulators implicated in anaerobic metabolism and denitrification that likely mediate this developmental transition. Our overall goal is to define the mechanisms underlying redox balancing for cells in biofilms, the conditions that determine their utilization and their spatiotemporal integration during biofilm development. We hypothesize that a complex regulatory network controls metabolic and morphogenetic responses to the conditions in P. aeruginosa biofilms such that intracellular redox homeostasis is maintained. We will map electron acceptor availability and intra- and extracellular redox potentials in developing colonies (Aim 1). We will verify that phenazine biosynthesis/ reduction and denitrification pathways engage in regulatory cross-talk and delineate the regulatory cascades controlling their activity in biofilms (Aim 2). Finally, we will characterize the components required for colony structure determination and investigate PAS domain protein-dependent mechanisms that link electron acceptor availability and community behavior (Aim 3). These multiple lines of inquiry will reveal the 3D distribution of exogenous and endogenous electron acceptors and their effects on bacterial physiology within specific microdomains of P. aeruginosa colonies. The means by which P. aeruginosa integrates environmental cues to support growth and survival in a crowded structure may be broadly applicable to many bacterial pathogens and have the potential to inform future therapeutic considerations.